Protein toxins are determinants of bacterial virulence, vaccine targets, and tools for development of new therapeutic approaches. Diphtheria toxin is a protein that crosses cellular membranes in order to inactivate protein synthesis. The goal of this project is to determine the structure of diphtheria toxin in its membrane-inserted state, and the mechanism by which its catalytic A chain translocates across membranes. Fluorescence methods to analyze toxin structure were developed in the previous grant period. These methods: differentiate residues exposed to solution from those buried in the membrane; measure the depth of membrane-buried residues; differentiate residues facing the aqueous solution outside the membrane from those facing the internal aqueous solution; detect oligomerization; and reveal the size of toxin induced pores. Using these methods it was established that the T domain of the toxin, which is critical for translocation, can exist both in states in which helices 8 and 9 partially penetrate into membranes and another in which they penetrate fully. In the next grant period, the topography of membrane-inserted mutant toxins will be examined with single fluorescent residues introduced by site-directed mutagenesis and chemical labeling. The complete topography of the helices of will be determined in both conformations of membrane-inserted T domain. Next, the topography of membrane-inserted A chain and membrane-inserted A chain-T domain complex will be determined. Residues critical for formation of the fully inserted state will be identified using the fluorescence techniques to examine the effect of amino acid substitutions introduced by site-directed mutagenesis. In addition, the implications of our observation that membrane-associated T domain interacts specifically with proteins that partly unfold and form the so- called "molten globule" state will be explored. Since the A chain also can partly unfold under physiological conditions, it is possible the T domain functions like a membrane-inserted chaperone of partly unfolded proteins. To test this idea, the nature of T domain interactions of with molten globule proteins will be compared to those with the A chain. Finally, the action of compounds we found to inhibit the pores induced by the toxin (and isolated T domain) in membranes will be examined, as well as whether they can block pores formed by other toxins. Thus, in addition to a better understanding of diphtheria toxin, these studies will yield new approaches for studying the membrane protein structure, insights into protein translocation across membranes, and clues to novel therapeutic agents.